肠三叶因子在大肠杆菌中的表达、纯化及其抗体制备

目的:获得肠三叶因子(ITF)的原核表达产物及抗rITF抗体,为深入研究ITF的作用机制及其受体研究奠定基础。方法:常规提取人小肠组织总RNA,用RT-PCR获得ITF编码基因片段,克隆至质粒pET32a获得原核表达栽体,双酶切和测序后转化至Origami B(DE3)用IPTG诱导表达,优化条件获得最大表达产量;用SDS-PAGE、Western blot鉴定表达产物,亲和层析纯化获得的重组蛋白rITF皮下多点注射家兔,制备多克隆抗体,并用此抗体进行大鼠肠组织免疫组化研究。结果:测序证实PCR扩增获得ITF全长基因序列与基因文库中的完全一致,将该基因片段正确插入表达载体pET32a中、优化表达条件后,重组蛋白的表达量达到50mg/L;Western blot证明重组蛋白具有良好的抗原性和特异性;通过Ni-NTA亲和层析、超滤离心后,得到90%纯度的蛋白;收集兔血清,纯化后获得特异性良好的ITF抗体,免疫组化染色肠组织显示ITF表达的部位定位于杯状细胞。结论:成功构建了表达载体pET32a-ITF,在大肠杆菌中表达并纯化获得纯度较高的rITF,并获得了生物活性较高的ITF抗体,ITF主要在肠道杯状细胞分泌表达。

Expression and Purification of Human Intestinal Trefoil Factor in Escherichia Coli and Preparation of Its Antibody

Objective:To express the gene fragment encoding for ITF in Escherichia coli and prepare the antibody,and also to lay the foundations for further study of its Mechanism and pharmaceutical function.Method:The gene fragment which encoding for mature peptide of ITF was obtained by RT-PCR,then inserted into the MCS of the expression vector pET32a to construct the recombinant vec- tor.After double enzyme digestion and gene sequencing,the recombinant vector was transformed into E.coli Origami B(DE3),and rhITF was expressed by IPTG induction,rhITF was purified by Ni-NTA affinity chromatography,determined by SDS-PAGE and West- ern blot analysis.The antibody to ITF was prepared by injection the purified recombinant protein to rabbits,and then was used to Im- munohistochemistry study of rat's small intestine Result:Sequencing results showed that the gene fragment obtained by RT-PCR was consistent with the reference sequence(Genl3ank Accession No.NM003226),and then it was successfully inserted into pET32a to con- struct the recombinant vector.The rhITF was expressed at a concentration of 50 mg/L.After purification,the purity of recombinant pro- tein was above 70%.The result of western blot analysis showed that rhITF had fine antigenicity and specificity.The ITF antibody collect- ed and purified from the rabbits serum was confirmed to have good specificity,and expressed in the goblet cell by immunohistochemistry study.Conclusion:The expression vector pET32a-ITF was constructed and expressed in E.coli successfully,and the recombinant protein rITF and its antibody was also purified in this study.ITF expressed in small intestine goblet cell.